Protein tyrosine phosphatases (PTPs) play an important role in intracellular signal transduction mediating cell growth, cell differentiation, transcription and metabolism (Neel, B. G. et al., Curr. Opin. Cell Biol. 9, 193-204, 1997; Zhang, Z.-Y. Annu. Rev. Pharmacol. Toxicol. 41, 209-234, 2002). Members of PTP family have been known to have a preserved active site where active cysteine residues reside. PTP family is composed of over 120 proteins, and it is divided into two sub-groups according to their target proteins and structural characteristics. The first sub-group is characterized by mediating dephosphorylation of pTyr only, and the second sub-group is characterized by mediating dephosphorylation of not only pTyr but also pSer/pThr.
Among them, phospatase of regenerating liver (referred as “PRL” hereinafter) is divided into three subtypes; PRL-1˜3, and has prenylated C-terminal (Diamond, R. H. et al., Mol. Cell. Biol. 14, 3752-3762, 1994). PRL-1 is an immediate early gene which is found in mouse liver and was cloned foremost (Mohn, K. L. et al. Mol. Cell. Biol. 11, 381-390, 1991). Then, PRL-2 and PRL-3 were found by homologous sequence search. The said PRL proteins include PTP motif sequence, which does not have homology with earlier PTP protein sequences, though. The modified phenotype of epithelial cells is resulted from the over-expression of PRL-1 or PRL-2, and cells transfected with those proteins turn into tumor cells in a nude mouse (Cates, C. A. et al., Cancer Lett. 110, 49-55, 1996). PRL-3 is remarkably over-expressed during the metastasis of colorectal cancer to the liver. However, the protein is not expressed in non-metastatic cancer cells or in general colorectal epithelial cells (Saha, S. et al., Science 294, 1343-1346, 2001). Amplification of PRL-3 gene is also found in the regions of metastasis in patients with different cancers.
Pentamidine, known as a PRL protein inhibitor, definitely inhibits the growth of WM9 human melanoma in a nude mouse (Manas, M. K. et al. Mol. Cancer. Therapeutics 1, 1255-1264, 2002). The over-expressions of PRL-1 and PRL-3 are involved in metastasis by accelerating migration and infiltration of cells (Zeng, Q. et al. Cancer Research 63, 2716-2722, 2003).
PRL proteins have preserved CAAX sequence (C: cysteine, A: aliphatic amino acid, X: amino acid) at C-terminal for prenylation. Protein prenylation, helping migration of a protein to cell membrane, plays an important role in intracellular signal transduction, along with receptor complex binding Ras and G-protein (Silvius, J. R., J. Membrane Biol. 190, 83-92, 2002; Takida, S. et al., J. Biol. Chem. 278, 17284-17290, 2003). Prenylated PRL proteins are transferred to plasma membrane and early endosome. At this time, when a farnesyltransferase inhibitor is treated thereto or mutation is induced in prenylated area, the PRL proteins are biased to nucleus (Zeng, Q. et al., J. Biol. Chem. 275, 21444-21452, 2000). According to a recent report, PRL-1 is biased cell cycle dependently (Wang, J. et al., J. Biol. Chem. 277, 46659-46668, 2002). That is, in nondividing cells, PRL-1 protein is biased to ER (endoplasmic reticulum) farnesylation dependently. In the mean time, in dividing cells, PRL-1 protein is biased to centrosome and spindle fiber, suggesting that PRL-1 plays an important role in cell division procedure by regulating the growth of spindle fiber.
Although accumulated test results on the effect of PRL protein on cell proliferation and tumorigenesis provide the possibility of a PRL protein inhibitor as a promising anticancer agent, concrete intracellular mechanism of PRL protein has not been disclosed, yet. It was recently reported that PRL-3 has NMR structure (Kozlov, G. et al., J. Biol. Chem. Epub M312905200, 2004), but it is an open structure of WPD loop, meaning that residues of an active site are rearranged from their enzyme catalytic site. Thus, exactly speaking, it is not an active structure of PRL enzyme.
For the purpose of elucidating detailed mechanism of PRL enzyme on the recognition of substrate and the regulation of activity, the present inventors provide a method of crystallization of the protein including the steps of mass-expressing human originated PRL-1 protein in E. coli transformant and purifying the expressed proteins, and also provide the resultant protein crystals. The present inventors completed this invention by confirming the tertiary active structure of PRL-1 protein at the resolution of 2.7 Å. It was confirmed that PRL-1 protein has a tertiary structure having 5 strands of beta-sheet surrounded by 6 alpha-helices and well arranged active site with closed P-loop, and monomers form a trimer through farnesylation site in the C-terminus of the PRL-1 protein. Thus, intracellular migration and membrane localization can be achieved.